To determine the levels of endotoxin, which is a major component of outer membrane of Gram-negative bacteria, and protein in the atmosphere in Sasebo, Japan, we measured these biological materials in fine (aerodynamic diameter ≤2.5 µm) and coarse (≥2.5 µm) particles collected for 81 weeks (September 2014 to May 2016). The monthly concentrations ( i.e. , the mean value of weekly concentrations for each month) of endotoxin were higher in coarse particles than in fine particles. Fluctuations in monthly endotoxin concentrations were large in both fine (0.0005–0.0208 EU/m³) and coarse (0.0032–0.1164 EU/m³) particles. Furthermore, the endotoxin concentrations in coarse particles were highest in October 2014 and 2015 as well as September 2014 (0.0407–0.1164 EU/m³). However, the monthly protein concentrations were higher in fine particles than in coarse particles. Compared to the endotoxin concentrations, the fluctuations in the monthly protein concentrations were smaller in both coarse and fine particles. To our knowledge, this study is the first to report long-term atmospheric concentrations of endotoxin and protein in Japan. Since the endotoxin concentrations in coarse particles were positively associated with the concentrations of Na⁺ and Cl⁻, it suggests the involvement of Gram-negative bacteria from seawater to the endotoxin levels in the atmosphere. For fine particles, the protein concentrations were positively associated with the concentrations of particles, NO₃⁻ and SO₄²⁻. These results suggest that combustion of organic materials, such as biomass burning, may be a contributor to atmospheric protein during this study period.