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The reduction of bacteria and biofilm formation is important when designing surfaces for use in industry. Molybdenum disulfide surfaces (MoS2SUR) were produced using MoS2particle (MoS2PAR) sizes of 90 nm, 2 μm, and 6 μm containing MoS2PARconcentrations of 5%, 10%, 15%, and 20%. These were tested to determine the efficacy of the MoS2SURto impede bacterial retention and biofilm formation of two different types of bacteria, Staphylococcus aureusand Pseudomonas aeruginosa. The MoS2SURwere characterized using Fourier transform infrared spectroscopy, ion-coupled plasma atomic emission spectroscopy, scanning electron microscopy, optical profilometry, and water contact angles. The MoS2SURmade with the smaller 90 nm MoS2PARsizes demonstrated smaller topographical-shaped features. As the size of the incorporated MoS2PARincreased, the MoS2SURdemonstrated wider surface features, and they were less wettable. The increase in MoS2PARconcentration within the MoS2SURgroups did not affect the surface topography but did increase wettability. However, the increase in MoS2PARsize increased both the surface topography and wettability. The MoS2SURwith the smaller topographical-shaped features influenced the retention of the S. aureusbacteria. Increased MoS2SURtopography and wettability resulted in the greatest reduction in bacterial retention, and the bacteria became more heterogeneously dispersed and less clustered across the surfaces. The surfaces that exhibited decreased bacterial retention (largest particle sizes, largest features, greatest roughness, and most wettable) resulted in decreased biofilm formation. Cytotoxicity testing of the surface using cell viability demonstrated that the MoS2SURwere not toxic against HK-2 cells at MoS2PARsizes of 90 nm and 2 μm. This work demonstrated that individual surface variables (MoS2SURtopographic shape and roughness, MoS2PARsize, and concentration) decreased bacterial loading on the surfaces, which then decreased biofilm formation. By optimizing MoS2SURproperties, it was possible to impede bacterial retention and subsequent biofilm formation.