Agro-industrial areas are frequently affected by various sources of atmospheric pollutants that have a negative impact on public health and ecosystems. However, air quality in these areas is infrequently monitored because of their smaller population compared to large cities, especially in developing countries. The Cauca River valley (CRV) is an agro-industrial region in southwestern Colombia, where a large fraction of the area is devoted to sugarcane and livestock production. The CRV is also affected by road traffic and industrial emissions. This study aims to elucidate the chemical composition of particulate matter fine mode (PM2.5) and to identify the main pollutant sources before source attribution. A sampling campaign was carried out at a representative site in the CRV region, where daily averaged mass concentrations of PM2.5 and the concentrations of water-soluble ions, trace metals, organic and elemental carbon, and various fractions of organic compounds (carbohydrates, n alkanes, and polycyclic aromatic hydrocarbons – PAHs) were measured. The mean PM2.5 was 14.4±4.4 µg m−3, and the most abundant constituent was organic material (52.7 % ± 18.4 %), followed by sulfate (12.7 % ± 2.8 %), and elemental carbon (7.1 % ± 2.5 %), which indicates the presence of secondary aerosol formation and incomplete combustion. Levoglucosan was present in all samples, with a mean concentration of (113.8±147.2 ng m−3), revealing biomass burning as a persistent source. Mass closure using the elemental carbon (EC) tracer method explained 88.4 % on PM2.5, whereas the organic tracer method explained 70.9 % of PM2.5. We attribute this difference to the lack of information of specific organic tracers for some sources, both primary and secondary. Organic material and inorganic ions were the dominant groups of species (79 % of PM2.5). OMprim and OMsec contribute 24.2 % and 28.5 % to PM2.5. Inorganic ions as sulfate, nitrate, and ammonia constitute 19.0 %, EC 7.1 %, dust 3.5%, particle-bounded water (PBW) 5.3 %, and trace element oxides (TEOs), 0.9 % of PM2.5. The aerosol was acidic, with a pH of 2.5±0.4, mainly because of the abundance of organic and sulfur compounds. Diagnostic ratios and tracer concentrations indicate that most PM2.5 was emitted locally and had contributions of both pyrogenic and petrogenic sources, that biomass burning was ubiquitous during the sampling period and was the main source of PAHs, and that the relatively low PM2.5 concentrations and mutagenic potentials are consistent with low-intensity, year-long biomass burning (BB) and sugarcane pre-harvest burning in the CRV.