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Human pathogens can survive and grow in hot springs. For water quality assessment, Escherichia coli or Enterococci are the main thermotolerant enteric bacteria commonly used to estimate the load of pathogenic bacteria in water. However, most of the environmental bacteria are unculturable thus culture methods may cause bias in detection of most pathogens. Illumina sequencing can provide a more comprehensive and accurate insight into environmental bacterial pathogens, which can be used to develop better risk assessment methods and promote public health awareness. In this study, high-throughput Illumina sequencing was used to identify bacterial pathogens from five hot springs; Maiwooi, Akwar, Garbanabra, Elegedi and Gelti, in Eritrea. Water samples were collected from the five hot springs. Total community DNA was extracted from samples using the phenol-chloroform method. The 16S rRNA gene variable region (V4-V7) of the extracted DNA was amplified and library construction done according to Illumina sequencing protocol. The sequence reads (length >200 bp) from Illumina sequencing libraries ranged from 22,091 sequences in the wet sediment sample from Garbanabra to 155,789 sequences in the mat sample from Elegedi. Taxonomy was assigned to each OTU using BLASTn against a curated database derived from GreenGenes, RDPII, SILVA SSU Reference 119 and NCBI. The proportion of potential pathogens from the water samples was highest in Maiwooi (17.8%), followed by Gelti (16.7%), Akwar (13.6%) and Garbanabra (10.9%). Although the numbers of DNA sequence reads from Illumina sequencing were very high for the Elegedi (104,328), corresponding proportion of potential pathogens very low (3.6%). Most of the potential pathogenic bacterial sequences identified were from Proteobacteria and Firmicutes. Legionella and Clostridium were the most common detected genera with different species. Most of the potential pathogens were detected from the water samples. However, sequences belonging to Clostridium were observed more abundantly from the mat samples. This study employed high-throughput sequencing technologies to determine the presence of pathogenic bacteria in the five hot springs in Eritrea.