Concentrated polymer brushes (CPBs) are known to suppress biofouling phenomena, such as protein adsorption and cell adhesion. However, a cumbersome process is needed for their synthesis. Here, we report a simple and versatile method for fabricating nonbiofouling coatings that uses well-defined bottlebrushes instead of CPBs. First, a macroinitiator, poly[2-(2-bromoisobutyryloxy)ethyl methacrylate] (PBIEM), was synthesized by reversible addition-fragmentation chain transfer polymerization. Then, poly[poly(ethylene glycol) methyl ether methacrylate] was grafted from PBIEM through atom transfer radical polymerization to form well-defined bottlebrushes. By controlling the graft chain length, two types of bottlebrushes could be prepared, namely those with a semi-dilute polymer brush (SDPB) structure or a CPB structure on the surface of the outermost layer. Crosslinked films of the bottlebrushes were prepared on silicon wafers by spin-coating and subsequent radical coupling. Importantly, the CPB-type bottlebrush films showed significantly better nonbiofouling characteristics than those of the SDPB-type bottlebrush films.