Investigating fine particulate matter (PM2.5) toxicity is crucial for health risk assessment and pollution control. This study explores the developmental toxicity of two PM2.5 sources: standard reference material 2786 (NIST, USA) and PM2.5 from Chakri Naruebodindra Medical Institute (CNMI, Thailand) located in the Bangkok Metropolitan area. Zebrafish embryos exposed to these samples exhibited embryonic mortality, with 50% lethal concentration (LC50) values of 1476 µg/mL for standard PM2.5 and 512 µg/mL for CNMI PM2.5. Morphological analysis revealed malformations, including pericardial and yolk sac edema, and blood clotting in both groups. Gene expression analysis highlighted source-specific effects. Standard PM2.5 downregulated sod1 and cat while upregulating gstp2. Inflammatory genes tnf-α and il-1b were upregulated, and nfkbi-αa was downregulated. Apoptosis-related genes bax, bcl-2, and casp3a were downregulated. CNMI PM2.5 consistently downregulated all examined genes. These findings underscore PM2.5 source variability's significance in biological system impact assessment, providing insights into pollutant-gene expression interactions. The study emphasizes the need for source-specific risk assessment and interventions to address PM2.5 exposure's health impacts effectively.