Objectives: The main objective of this study is to assess the levels of hazardous substances in floor dust in primary schools located in the city of Busan in Korea. Materials and Methods: An on-site investigation of three primary schools was performed between April and May 2013. The hazardous substances measured in this study were 14 heavy metals (Cu, Fe, Pb, Mn, Ni, Zn, Cr, Cd, As, Al, Sn, Co, Mo and Si) and the biological agents were bacteria, fungi and endotoxin). Results: Among the heavy metals, Cd, Co, Pb and Cr were not detected in the floor dust from the three primary schools. The mean levels of other heavy metals were as follows: $20({\pm}10)ng/cm^2$ for As, $30({\pm}20)ng/cm^2$ for Al, $5({\pm}4)ng/cm^2$ for Sn, $20({\pm}20)ng/cm^2$ for Mo, $1,340({\pm}620)ng/cm^2$ for Si, $110({\pm}100)ng/cm^2$ for Cu, $240({\pm}50)ng/cm^2$ for Fe, $30({\pm}30)ng/cm^2$ for Mn, $10({\pm}10)ng/cm^2$ for Ni, and $50({\pm}30)ng/cm^2$ for Zn. It was found that mean concentrations of bacteria, fungi and endotoxin in the floor dust of primary schools were $4.7{\time}10^7({\pm}2.2{\time}10^7)cfu/cm^2$, $6.3{\time}10^6({\pm}6.4{\time}10^6)cfu/cm^2$, and $8,140({\pm}5,801)EU/cm^2$, respectively. The predominant species identified in the floor dust of the primary schools were Pseudomonas spp. for bacteria and Penicillium spp.,Cladosporidium spp.,and Aspergillus spp. for fungi, which would be somewhat similar to the microbial distribution pattern of other general environments. Conclusions: Based on the results obtained from this study, the levels of heavy metals, microbes and endotoxin distributed in the floor dust of primary school were higher than those reported for other general facilities. Thus, preventive measures should be prepared for the health care of children.