Conocarpus lancifolius leaves were observed to have typical symptoms of fungal leaf spots in botanical gardens and various nurseries, mainly during the winter and rainy seasons of 2022 and 2023. Leaves of infected plants showed round to irregular grayish-white and dark brownish to black spots with a yellow halo and central whitish color appearance that were variable in size, ranging from 0.5 to 1.5 mm in radius. After processing the samples and purification of the fungus, morphological characteristics such as its color, mycelium pattern, and spore shape were used to identify it as belonging to the genus Alternaria. Following morphological identification pathogenicity test was performed according to Koch’s postulates, proving that the pathogenic fungus Alternaria burnsii was associated with C. lancifolius causing leaf spots. The CTAB method was used to extract DNA to perform molecular characterization and further species-level identification. The fungal DNA was amplified using the ITS and GAPDH genes. Sequencing was done by Macrogen, Korea. A phylogenetic tree was constructed against both genes (ITS and GADPH) by using the sequence of 20 isolates; 19 belong to the Alternaria family and 1 is an outgroup. The suspected isolate with accession no OQ689862, and OQ910482 showed 99.48% and 100% similarity with OP985911 and MN718663, respectively. After submitting sequences to NCBI BanKit, the following accession numbers were assigned against the ITS and GAPDH gene sequences: OQ689862 and OQ910482, respectively. After molecular and morphological characterization, it was determined that Alternaria burnsii was isolated and associated with the leaf spots of the C. lancifolius plant by satisfying Koch’s postulates. According to our research, this is the first report of the leaf spot of C. lancifolius caused by A. burnsii. It is suspected that in the future, C. lancifolius will be infected by other pathogenic microbes. Therefore, to control the disease spread, highly effective management strategies should be adopted.