diff --git a/CHANGELOG.md b/CHANGELOG.md
index 66a77dd299..c4e97a5c88 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -20,6 +20,7 @@
 
 ### Template
 
+- New pre-commit hooks (large files, merge conflicts) in pipeline template ([#3935](https://github.com/nf-core/tools/pull/3935))
 - switch to uv and prek for pipeline linting workflow ([#3942](https://github.com/nf-core/tools/pull/3942))
 
 ### Linting
diff --git a/nf_core/pipeline-template/.hooks/block_pipeline_outdir.sh b/nf_core/pipeline-template/.hooks/block_pipeline_outdir.sh
new file mode 100755
index 0000000000..fbd2419181
--- /dev/null
+++ b/nf_core/pipeline-template/.hooks/block_pipeline_outdir.sh
@@ -0,0 +1,30 @@
+#!/usr/bin/env bash
+# This hook is used to block commits if they include staged files inside a directory
+# which also contains a subdirectory called `pipeline_info`. The purpose of this is to
+# prevent users from inadvertently committing output from pipeline test runs inside the
+# development directory.
+
+set -e
+
+# list staged files
+staged_files="$(git diff --cached --name-only)"
+
+status=0
+
+for file in $staged_files; do
+    file_dir=$(dirname "$file")
+
+    # Walk up the directory tree and check if the current directory contains a subdirectory called `pipeline_info`
+    # or the staged file is itself inside a directory called `pipeline_info`.
+    while [ "$file_dir" != "." ] && [ "$file_dir" != "/" ]; do
+        if [ $(basename "$file_dir") == "pipeline_info" ] || [ -d "$file_dir/pipeline_info" ]; then
+        echo "❌ Commit blocked: Please do not commit output from pipeline test runs to the pipeline code itself."
+        echo "Use 'git restore --staged <file>...' to remove the output files from the staging area before proceeding."
+        status=1
+        break
+        fi
+        file_dir=$(dirname "$file_dir")
+    done
+done
+
+exit "$status"
diff --git a/nf_core/pipeline-template/.pre-commit-config.yaml b/nf_core/pipeline-template/.pre-commit-config.yaml
index d06777a8f7..9ee191a0ef 100644
--- a/nf_core/pipeline-template/.pre-commit-config.yaml
+++ b/nf_core/pipeline-template/.pre-commit-config.yaml
@@ -25,3 +25,20 @@ repos:
               subworkflows/nf-core/.*|
               .*\.snap$
           )$
+      - id: check-added-large-files
+        args: [--maxkb=5000]
+        exclude: |
+          (?x)^(
+              .*ro-crate-metadata.json$|
+              .*\.snap$|
+              lib/nfcore_external_java_deps.jar$|
+              docs/.*\.(svg|pdf)$|
+              assets/.*$
+          )$
+      - id: check-merge-conflict
+  - repo: local
+    hooks:
+      - id: block-pipeline-outdir
+        name: Prevent committing output from pipeline test runs to the pipeline code itself
+        entry: ./.hooks/block_pipeline_outdir.sh
+        language: script
diff --git a/nf_core/pipelines/create/template_features.yml b/nf_core/pipelines/create/template_features.yml
index 0801f1ff75..63a019b3f9 100644
--- a/nf_core/pipelines/create/template_features.yml
+++ b/nf_core/pipelines/create/template_features.yml
@@ -438,6 +438,7 @@ code_quality:
         - ".prettierignore"
         - ".prettierrc.yml"
         - ".github/workflows/fix-linting.yml"
+        - ".hooks/block_pipeline_outdir.sh"
       short_description: "Use code linters"
       description: "The pipeline will include code linters and CI tests to lint your code: pre-commit, editor-config and prettier."
       help_text: |