Harbours are important for economic and social development of coastal areas but they also represent an anthropogenic source of emissions often located near urban centres and industrial areas. This increases the difficulties in distinguishing the harbour contribution with respect to other sources. The aim of this work is the characterisation of main sources of PM_2.5 acting on the Brindisi harbour–industrial area, trying to pinpoint the contribution of in-port ship emissions to primary and secondary PM_2.5. Brindisi is an important port-city of the Adriatic Sea considered a hot-spot for anthropogenic environmental pressures at National level. Measurements were performed collecting PM_2.5 samples and characterising the concentrations of 23 chemical species (water soluble organic and inorganic carbon; major ions: SO₄^2 −, NO₃⁻, NH₄⁺, Cl⁻, C₂O₄^2 −, Na⁺, K⁺, Mg^2 +, Ca^2 +; and elements: Ni, Cu, V, Mn, As, Pb, Cr, Sb, Fe, Al, Zn, and Ti). These species represent, on average, 51.4% of PM_2.5 and were used for source apportionment via PMF. The contributions of eight sources were estimated: crustal (16.4 ± 0.9% of PM_2.5), aged marine (2.6 ± 0.5%), crustal carbonates (7.7 ± 0.3%), ammonium sulphate (27.3 ± 0.8%), biomass burning-fires (11.7 ± 0.7%), traffic (16.4 ± 1.7 %), industrial (0.4 ± 0.3%) and a mixed source oil combustion–industrial including ship emissions in harbour (15.3 ± 1.3%). The PMF did not separate the in-port ship emission contribution from industrial releases. The correlation of estimated contribution with meteorology showed directionality with an increase of oil combustion and sulphate contribution in the harbour direction with respect to the direction of the urban area and an increase of the V/Ni ratio. This allowed for the use of V as marker of primary ship contribution to PM_2.5 (2.8% +/− 1.1%). The secondary contribution of oil combustion to non-sea-salt-sulphate, nssSO₄^2 −, was estimated to be 1.3 μg/m³ (about 40% of total nssSO₄^2 − or 11% of PM_2.5).