Systematic disinfection of the stethoscope diaphragm is required to ensure that it does not act as a vector for cross-transmission of health-related diseases. Thus, an antimicrobial latex film could be used as a cover to inhibit pathogenic bacteria from growing on its surface. The aim of this work is to determine the antimicrobial activity and mechanical properties of antimicrobial natural rubber (NR) latex films with different types of antimicrobial agents (mangosteen peel powder (MPP), zinc oxide nanoparticles (ZnO NP), and povidone-iodine (PVP-I)). The antimicrobial loading was varied from 0.5, to 1.0, and 2.0 phr to monitor the effective inhibition of Gram-negative bacteria and fungi growth. For MPP and PVP-I antimicrobial agents, a loading of 2.0 phr showed good antimicrobial efficacy with the largest zone of inhibition. Simultaneously, ZnO NP demonstrated excellent antimicrobial activity at low concentrations. The addition of antimicrobial agents shows a comparable effect on the mechanical properties of NR latex films. In comparison to control NR latex film (29.41 MPa, 48.49 N/mm), antimicrobial-filled films have significantly greater tensile and tear strengths (MPP (33.84 MPa, 65.21 N/mm), ZnO NP (31.79 MPa, 52.77 N/mm), and PVP-I (33.25 MPa, 50.75 N/mm). In conclusion, the addition of antimicrobial agents, particularly ZnO NP, can be a better choice for NR latex films because they will serve as both an activator and an antimicrobial. In a clinical context, with regard to frequently used medical equipment such as a stethoscope, such an approach offers significant promise to aid infection control.