In recent year, the chemistry of ferrocene and the design of new compounds containing the ferrocene unit, has received a surge in interest, owing to their utility in many fields such as organic synthesis, catalysis and biotechnology material. In the framework, of attending a new multifunctional ferrocenyl derivatives, two precurssors namely DMHF and a carbaldehyde ferrocene functionalized in 1,2 position by thiophosphine function were fully characterized by HRMS, IR, NMR ¹H, ¹³C, and UV spectroscopy, then were in vitro tested for antibacterial activity against two gram+ (Staphylococcus aureus, Bacillus sp), three gram- (E.coli, Klebsiella pneumonia, Morganella morganii )referential strains and Penicillium Sp fungi using disk diffusion method on solid medium MH and Sabouraud respectively. Obtained results were compared to antibiogram and reveled an interesting antimicrobial potent varying from 7 to 10mm of inhibition for DMHF and 7 to 15mm of inhibition for 1,2 dithiophosphine ferrocene compounds, which indicates mostly an improvement of antimicrobial activity with chiral substitution of ferrocene moiety with thiophosphine function, this encourage future investigations on chirality and substitution role in the improvement of antimicrobial activity.