A method was developed for effect-directed analysis (EDA) of the root extract of Pimpinella saxifraga L. High-performance thin-layer chromatography (HPTLC) was hyphenated with microchemical, biochemical, and biological assays as well as electrospray ionization– mass spectrometry (ESI–MS). This HPTLC–UV/Vis/FLD– EDA–MS method directly pointed to multi-potent compounds in the P. saxifraga L. root extract. 2,2-Diphenyl-1-picrylhydrazyl radical scavengers, acetylcholinesterase inhibitors, estrogen-effective compounds, antimicrobials against Gram-positive Bacillus subtilis bacteria, and Gram-negative Aliivibrio fischeri bacteria were discovered in the root extract. A first targeted characterization of four unknown multi-potent compounds was performed by HPTLC–ESI–MS and microchemical derivatizations. This highly streamlined effect-directed profiling is recommended for a fast and cost-efficient natural product search.