Due to perpetual increasing antimicrobial resistance, functionally substituted alicyclic compounds are envisioned as probable antimicrobial agents of future. From screening of in house library of compounds, here we report the antimicrobial properties of three novel functionally substituted monocyclic and spirocyclic cyclohexane derivatives involving ethyl-3 (allylamino)-9,9-dimethyl- 7,11-dioxo-1,5-diphenylspiro[5.5]undec-2-en-2-carboxylate (I), ethyl-4,6-diphenyl-2- dicyanomethylene cyclohex-3-ene 1-carboxylate (II) and ethyl-4-phenyl-6-(4-chlorophenyl)-2- dicyanomethylenecyclohex-3-ene 1-carboxylate (III).Initially, these compounds were screened for in vitro antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi by agar well diffusion method. In second step, minimum inhibitory concentration of test compounds was determined against bacterial species by using resazurin microplate assay. All the tested compounds exhibited variable antimicrobial properties against various test cultures. All the compounds showed stronger antimicrobial activity against Gram-negative bacteria as compared to Gram-positive bacteria and fungi. Compound III was found to be the most effective compound. These results demonstrate the potential antimicrobial properties of mono and spiro cyclohexane derivatives and accentuate the need of in vivo trials for their application at clinical level.