The widespread use of antibiotics in medicine and agriculture has significantly accelerated the emergence rate of bacterial infections showing multiple antibiotic resistance. Since resistance to conventional antibiotics is developed rather quickly, designing alternative antimicrobial drugs with other mechanisms underlying their effects on bacteria is a promising. The enzymes possessing bactericidal activity may be one option for such antibacterial agents. The study aimed to produce the combination recombinant protein-based products active against bacteria and their biofilms. Soluble forms of five recombinant proteins were produced using the genetic engineering approaches. Two of these have a bacteriolytic effect (endolysins LysK and PM9 from the Staphylococcus aureus bacteriophages), the other are capable of disrupting extracellular DNA matrix in biofilms (two nonspecific nucleases NucA, as well as the DNA-specific deoxyribonuclease I). It has been shown that natural endolysin PM9 with the truncated catalytic domain shows 4 times lower bacteriolytic efficacy compared to the full-size LysK version. Comparative analysis revealed 1.5–2 timed higher efficacy of nonspecific nucleases in terms of bacterial biofilm disruption compared to the DNA-specific deoxyribonuclease I. It has been shown that simultaneous use of endolysins and nucleases has a synergistic antibacterial effect and disrupts biofilms of the pathogenic bacterium Staphylococcus aureus. The findings show the prospects of developing the recombinant protein-based antibacterial drugs.