Background: Naegleria fowleri infection is rare and fatal with case fatality rate of 100%. Although many years after the first case of Naegleria was reported in Karachi, few integrated studies have been performed. This study aimed to identify the epidemiological characteristics, trends, risk factor analysis and environmental assessment of Southern Pakistan. Methods: Medical records were reviewed, and active case identification was carried out. The data were collected on sociodemographic information, clinical features, and risk factors on semi-structured questionnaire from June 6, 2023 to December 31, 2023. An analytical study, trend analysis and environmental assessment were performed. Pumping stations were investigated for chlorination and water samples were sent for analysis. Data were analysed by via Epi-Info and SPSS software. Results: A total of 101 cases were reported; 84% (85) were males. The average age was 29 years. Frequent symptoms were fever (100%), vomiting (100%), and coma (54%) followed by fits (34%) and confusion (32%). Among the cases, students (n=18), engineers (n=14) and shopkeepers (n=13) were highly infected. An analytical study revealed that people performing ablution daily (OR 3.7, 95% CI 1.6-11.7), swimming (OR 3.5 95% CI 1.1-11.5), taking daily baths (OR 7.3, 95% CI 2.3-23.9), performing religious rituals (OR 4.4 95% CI 1.4-13.5) and areas supplied by pumping station A (OR 4.5, 95% CI 1.1-17) were more prone. The case trends showed peaks in the summer season, which were correlated with the environmental temperature and humidity. A lack of chlorination was reported at the pumping station as the water samples have presented high levels of coliform colonies. Conclusions: Naegleria Fowleri is a major public health problem in the southern Pakistan. As per the WHO recommendation maintenance of the proper chlorination and water distribution system is required. Advocacy to communities and risk communication during peak seasons are mandated.