Introduction <p>Cement plants use refuse-derived fuel (RDF) as an important energy source in cement production.</p> Objective <p>The aim of the study was to characterize bacterial contamination of RDF and air at workplaces related to their combustion process.</p> Methods <p>The study was carried out in 4 cement plants in Poland. Bioaerosol sampling covered different stages of technological process of fuel combustion. Fourteen RDF samples were also collected. Quantitative and qualitative analysis of bacteria was performed using API biochemical tests.</p> Results <p>The median bacterial concentration found in RDF samples was at the level of 4.2 × 10⁶&#xa0;CFU&#xa0;g⁻¹ (1.1 × 10⁵–2.9 × 10¹²&#xa0;CFU&#xa0;g⁻¹). Median concentration values of airborne bacteria amounted to 1.9 × 10⁴&#xa0;CFU&#xa0;m⁻³ (1.9 × 10²–1.1 × 10⁵&#xa0;CFU&#xa0;m⁻³). Concentrations exceeded the threshold limit values for bacteria in the loader cabin (1.1 × 10⁵&#xa0;CFU&#xa0;m⁻³, 1.1 × 10⁵–1.2 × 10⁵&#xa0;CFU&#xa0;m⁻³) and laboratory rooms (1.3 × 10⁴&#xa0;CFU&#xa0;m⁻³, 1.9 × 10²–1.1 × 10⁵&#xa0;CFU&#xa0;m⁻³). Bioaerosol concentrations significantly correlated with air temperature and relative humidity (<i>p</i> &lt; 0.001). Gram-positive bacilli, as well as Gram-negative rods i.e. <i>Enterobacter</i>, <i>Escherichia</i>, <i>Klebsiella</i>, and <i>Proteus</i> genera were widely present in bioaerosol samples. This study confirmed, the ubiquity of 21 bacterial species classified in risk group 2 according to Directive 2019/1833 in both bioaerosol and alternative fuel samples.</p> Conclusion <p>In conclusion, RDF significantly affect the air quality at workplaces in cement plants. Therefore, employers should consider implementing various technical, organizational, and personal measures to protect exposed workers.</p> Graphical Abstract <p></p>