Bacterial resistance is a major issue in the modern world, and Staphylococcus aureus is one of these well-known multi-resistant species. Staphylococcal infections are one of the leading causes of infection in humans and are becoming more challenging to treat by conventional methods. Endolysins, a novel class of antibacterial agents, are bacteriophage-encoded lytic enzymes capable of degrading peptidoglycan and thus able to kill bacteria. This study aimed to study endolysin LYSDERM-S (a variant of endolysin LysF1 optimized for heterologous expression in E. coli) and newly prepared thermally stabilized endolysin LYSDERM-T1 (with a mutation in the CHAP domain) both with (LYSDERM-US, LYSDERM-UT1) and without fused ubiquitin and determine its role in protein expression and antibacterial activity. The results showed that fused endolysin-ubiquitin proteins did not exceed the antimicrobial effect of endolysins alone, but cleaved endolysin-ubiquitin proteins possessed longer lasting antimicrobial effect than endolysin alone. The biobetter endolysin LYSDERM-T1 with higher thermal stability showed a prolonged antimicrobial effect. Further, we showed that ubiquitin alone possesses antimicrobial properties. Minimal inhibitory and bactericidal concentrations (MIC and MBC) were assessed and confirmed that ubiquitin is able to increase the antimicrobial potential of endolysins. Biobetter endolysins or endolysin-ubiquitin combinations could serve as an alternative to well-established antimicrobial therapy for methicillin-resistant S. aureus infections.