The chemical characterization of particulate matter (PM) 2.5 fraction was studied during a 1-year sampling campaign conducted at a site near Thessaloniki’s port area. PM2.5 collected samples were chemically analyzed for polycyclic aromatic hydrocarbons, minerals, and trace elements (Pb, Ni, Cu, V, Mn, Cr, Zn, Mg, K, Ti, Fe, Ca, and Al); water-soluble ions (Cl−, NO3 −, SO4 2−, K+, Na+, NH4 +, Mg2+, Ca2+); and organic and elemental carbon. The average annual PM2.5 concentration (66.0 μg/m3) was at the highest level compared with other studies reported for the same city but different sampling sites. The average daily sum of the measured concentration of polycyclic aromatic hydrocarbons (PAHs) was 12.76 ng/m3; this value decreased to 6.73 ng/m3 for the warm period and reached the value of 19.8 ng/m3 for the cold period. The average concentration of benzo[a]pyrene during the sampling period was 0.75 ng/m3, which is below the European Union limit value of 1.0 ng/m3. The ionic content comprised, on average, 22.6 % of the PM2.5 mass, with sulfate and ammonium being the most abundant species (31 and 26 %, respectively, of measured ions during the whole sampling period). The annual mean concentrations of organic carbon (OC) and elemental carbon (EC) were 10.5 ± 6.3 and 2.3 ± 1.5 μg/m3, respectively. The OC/EC ratio ranged from 1.6 to 9.9, suggesting that there is a significant influence of residential wood burning for heating as well as ship and vehicle emissions to the sampling area. Finally, the elemental composition of associated PM2.5 was dominated by Ca, Fe, and Al. Although conclusions based only on PM2.5 measurements cannot entirely estimate all harbor sources’ contribution, there is evidence to support that port activities affect the city’s air quality and vice versa.