Yes if the WordPress detection tool tries to detect wp-content files like themes and plugin files it will return a 404 error and will return your site as not using WordPress if your have configured all other settings properly.
It also prevents automated attacks that target the default wordpress paths since the urls now return 404.
Ike explained very well to your question, thanks.
In addition, you should keep in mind that behind each detector is an algorithm which seek for certain fingerprints when creating a report. Many of those detectors rely also on urls / paths to identify themes and plugins.
