Abstract The evolution of multidrug resistance in bacteria instigates the decline in effectiveness of antibiotics. Nanoparticle-enzyme bioconjugates are a recent interest in countering biofilm-related infections due to their stability and targeted delivery. The synergistic effect of α-amylase and biosynthesized silver nanoparticles (AgNPs) towards biofilms remains less explored. In this study, we demonstrate the conjugation of α-amylase and AgNPs synthesized using the supernatant of Bacillus horikoshii AJM-A1 and the subsequent application against multidrug resistant biofilms. The biosynthesized AgNPs exhibited an absorbance peak at 400–470 nm which indicates the typical surface plasmon resonance of AgNPs. SEM-EDX analysis showed irregular and spherical nanoparticles with an average size of 13.47 ± 12 nm and high elemental silver. FTIR analysis revealed functional groups in the supernatant involved in reducing Ag+ to AgNP. The AgNPs were conjugated with α-amylase from Bacillus subtilis and the possible modification in the protein's structure was confirmed by fluorescence quenching, FTIR analysis, and absorbance measurements. The bactericidal and antibiofilm efficacies of the AgNPs and bioconjugates were tested against the multidrug-resistant strains Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) using varying concentrations (25, 50, 100, 200, 400, 800 μg/mL). The samples showed significant antibacterial activity down to the lowest concentration used and demonstrated significant inhibition of biofilm formation and dispersion of pre-formed biofilms in a dose-dependent manner. The bioconjugates exhibited higher efficiency than AgNPs and α-amylase when used individually against the biofilm-forming strains. This study demonstrates that AgNPs synthesized by alkaliphilic bacterium can be conjugated with α-amylase for potential application against resistant biofilms.