The global searchlight for sustainable alternative fuels to reduce emissions produced from the combustion of fossil fuels illuminates biofuels owing to their matching properties with fossil fuels. This is the impetus for this study which systematically examines the impact of neem biodiesel (NB) blends with pure diesel on the physical and chemical properties of particulate matter (PM) from diesel engines. Pure diesel (B0) and four fuel blends, namely, B5, B10, B15 and B20 are examined. The impact of NB blends on the physical and chemical properties of PM is studied using a single-cylinder, 4-stroke diesel engine. The PM captured directly from the diesel engine at two standard engine speeds is analyzed by physical microscopy techniques and chemical analyses. Comparing the results of gaseous emissions for B0 with those of B20, it is found that B20 decreases CO by 9.6% and 19.3% at low and high engine speeds, respectively, but increases NO. Regarding PM emission, in comparison to B0, B20 decreases particle sizes from 59.4 ± 8.5 nm to 42.8 ± 4.2 nm and 63.3 ± 8.1 nm to 43.7 ± 5.2 nm; opacities from 15.9% to 9.3% and 21.1%–11.4%; carbon contents from 66.53% to 44.53% and 72.53%–61.99%; and total carbon concentrations (total organic carbon and total inorganic carbon) from 3.6120 mg/L to 1.8435 mg/L and 2.5970 mg/L to 1.6002 mg/L at low and high engine speeds, respectively. Furthermore, B20 increases the unused oxygen content from 14.07% to 21.47% and 16.82%–18.42%; oxygen reactivity from 1.80 ± 0.08 to 2.75 ± 0.18 and 1.10 ± 0.20 to 1.35 ± 0.06; and volatile substances by 68.4% and 57.1% at low and high engine speeds, respectively. This study demonstrates that NB could be a potential alternative fuel for diesel engines regarding PM emissions, where B20 blend has the highest impact on PM properties, but it needs additional NOx mitigation strategies.