α-amylase is known to have antibiofilm activity against biofilms of both Gram positive and Gram-negative bacterial strains. Partially purified α-amylase from Bacillus subtilis was found to have inhibit biofilm formed by P. aeruginosa and S. aureus. The spectrophotometric and microscopic studies revealed that the antibiofilm efficacy of the working strain is greater than commercially purchased α-amylase. Response surface methodology (RSM) and artificial neural network (ANN) help to predict the optimum conditions pH 8, treatment time 6 h and enzyme concentration (200 µg/mL) for maximum biofilm eradication. This was confirmed by several in vitro experiments. Molecular docking interactions of α-amylase with the extracellular polymeric substances (EPS) of both P. aeruginosa and S. aureus indicate towards the existence of an efficient energy driven spontaneous process. Thus, this study highlights a combination of experimental and computational approach showing the naturally extracted α-amylase from B. subtilis having the potency of removing the biofilms of harmful bacterial strains involved in causing various nosocomial infections.