The high resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious global public-health concerns. This has stimulated interest in the research of bio-based therapeutics with limited resistance, namely, antimicrobial peptides (AMPs) or essential oils (EOs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control for comparison purposes. The biomolecules were initially screened for their antibacterial activity using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation through scanning electron microscopy (SEM) observations of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 µg/mL) to induce the same effects as the AMPs (500–7.8 µg/mL). Pexiganan was the most effective biomolecule, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were seen to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these biomolecules and allowed to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.