Abstract <p>New sulfur-containing Mannich bases derived from 1-(octylsulfanyl)hexan-2-ol, secondary amines (diethylamine, dibutylamine, piperidine, morpholine, hexamethyleneimine), and formaldehyde have been synthesized by three-component Mannich reaction and screened for antibacterial activity. The composition and structure of the synthesized compounds were confirmed by elemental analysis, IR and ¹H NMR spectroscopy, and mass spectrometry. The antimicrobial screening was performed using the disk diffusion assay. The synthesized Mannich bases showed high antimicrobial activity against Gram-positive and Gram-negative bacteria (<i>S. aureus</i>, <i>E. coli</i>, <i>P. aeruginosa</i>, and <i>K. pneumoniae</i>), as well as yeast-like fungi of the genus <i>Candida</i> in comparison with the reference antibacterial agents (rivanol, furacilin, carbolic acid, and chloramine). The resulting data allow the synthesized compounds to be recommended as candidate antimicrobial drugs.</p>