In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline substituents at 2nd position were synthesized and screed for biofilm inhibitory, antifungal and antibacterial activity. The result of biofilm inhibition of C. albicans suggested that compounds 5j (IC<sub>50</sub> value = 51.2 μM) and 5a (IC<sub>50</sub> value = 66.2 μM) possess promising antibiofilm inhibition when compared with the standard antifungal drug fluconazole (IC<sub>50</sub> = 40.0 μM). Two compounds 5a (MIC = 94.2 μg/mL) and 5f (MIC = 98.8 μg/mL) also exhibited good antifungal activity comparable to standard drug fluconazole (MIC = 50.0 μg/mL). The antibacterial screening against four strains of bacteria viz. E. coli, P. aeruginosa, B. subtilis, and S. aureus suggested their potential antibacterial activity and especially all the compounds except 5g were found more active than the standard drug ciprofloxacin against B. subtilis. To further gain insights into the possible mechanism of these compounds in biofilm inhibition through the agglutinin like protein (Als), molecular docking and molecular dynamics simulation studies were carried out. Molecular modeling studies suggested the clear role in inhibition of this protein and the resulting biofilm inhibitory activity.