New Six cationic Gemini surfactants (GS) with varying alkyl chain lengths and spacer properties were synthesized. The effects of alkyl chain length and spacer characters on antimicrobial activity were investigated. The first group of compounds was known as (2-2'-(ethane-1, 2-diylbis (oxy)) bis (N-(2-alkanamidoethyl)-N, N dimethyl-2-oxoethan-1-aminium)) dichloride, and it had three different alkyl chain lengths (CGSES12, 14 and 16). The general name for the second group was (N₁,N₁,N₃,N₃-tetramethyl-N₁,N₃-bis(2-alkanamidoethyl)propane-1,3-diaminium bromide) with three different alkyl chain lengths, designated as (CGSPS12, 14 and 16).The antimicrobial activity of each surfactant was evaluated by determining the minimum inhibitory concentration (MIC) and the zone of inhibition against six representative organisms: two Gram-positive bacteria; Staphylococcus aureus and Bacillus subtilis, two Gram-negative bacteria; Escherichia coli and Proteus vulgaris and two fungi; Aspergillus fumigates and Candida albicans. The study indicated that all newly synthesized surfactants exhibit antimicrobial activity, but the highest activity was determined for surfactants CGSES12, CGSPS12 i.e. those with the shortest alkyl chain. Remarkably, the lowest MIC value was obtained for CGSES12 against P.vulgaris (2 μg/mL). Obviously, the antibacterial activity did not correlate with the length of the spacer. However, the overall antibacterial activity increased with decreasing the length of the alkyl chain. Our data indicated that all synthesized cationic dimeric compounds satisfy the main requirement to be good surfactants, with potential applications as antimicrobials or disinfectants.