Primary aim of this in vitro study was to test the efficacy of daptomycin to eradicate staphylococcal biofilms on various orthopedic implant materials. Secondary aim was to quantitatively estimate the formation of staphylococcal biofilm. We tested six clinically important biomaterials: Cobalt chrome, pure titanium, grid-blasted titanium, porous plasma-coated titanium with/without hydroxyapatite, and polyethylene. Biofilms of S. aureus and S. epidermidis were formed on the samples and thereafter exposed to daptomycin. Samples were subsequently sonicated in order to detect dislodged biofilm bacteria and transferred to a microcalorimeter for real-time measurement of growth-related heat flow. Minimal biofilm eradication concentration (MBEC) was determined as the lowest concentration of daptomycin required to eradicate biofilm bacteria on the sample. Median MBEC of S. aureus biofilm on smooth metallic surfaces was lower than the rough metallic surfaces. In experiments with S. epidermidis, no pattern was seen in relation to the surface roughness. Regarding the quantitative estimation of staphylococcal biofilm formation on the sample, we found a significantly higher amount of biofilm growth on the rough surfaces than the smooth samples and polyethylene. In conclusion, the presented study showed that daptomycin could eradicate S. aureus biofilm at lower concentrations on the smooth surfaces compared to the rough surfaces, as well as polyethylene. In experiments with daptomycin against S. epidermidis biofilms, no pattern was seen in relation to the surface roughness. Furthermore, we demonstrated a faster detection of staphylococcal heat flow due to higher biofilm quantity on the rough surfaces compared to smooth samples and polyethylene.