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The present study shows the untargeted metabolite profiling and in vitro antibacterial, cytotoxic, and nitric oxide (NO) inhibitory activities of the methanolic leaves extract (MLE) and methanolic stem extract (MSE) of Erythroxylum mexicanum, as well as the fractions from MSE. Using ultra-high performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), a total of 70 metabolites were identified; mainly alkaloids in the MLE, while the MSE showed a high abundance of diterpenoids. The MSE fractions exhibited differential activity against Gram-positive bacteria. Notably, the hexane fraction (HSF) against Streptococcus pyogenes ATCC 19615 (MIC=62.5 μg/mL) exhibited a bactericidal effect. The MSE fractions exhibited cytotoxicity against all cancer cell lines tested, with selectivity towards them compared to a noncancerous cell line. Particularly, the HSF and chloroform fraction (CSF) showed the highest cytotoxicity against prostate cancer (PC-3) cells, with IC 50 values of 19.9 and 18.1 μg/mL and selectivity indexes of 3.8 and 4.2, respectively. Both the HSF and ethyl acetate (EASF) fractions of the MSE inhibited NO production in RAW 264.7 macrophages, with NO production percentages of 50.0 % and 51.7 %, respectively, at a concentration of 30 μg/mL. These results indicated that E. mexicanum can be a source of antibacterial, cytotoxic, and anti-inflammatory metabolites., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)