The aim of this study was to detect the pathogenic microorganisms that cause milk baby poisoning. Samples of powder milk purchased from local pharmacies were analysed by microbial and molecular methods. Recent advances in molecular techniques as 16S rDNA gene and sequencing were used as an identification of an unknown microorganism that requires comparison of its characteristics with data obtained for example from 16S rDNA gene. DNA of ten bacterial isolates subjected to specific PCR to amplify about 350 bp from 16S rDNA gene revealed the band in the right molecular weight with all the bacterial samples. RFLP was carried out by using two enzymes (MSP 1 and Hae 3). The results showed high similarity between all samples, where MSP 1 gave two bands at molecular size of 230 and 120 bp, respectively, and high similarity between all samples, where Hae 3 gave two bands at molecular size of 210 and 140 bp, respectively, which confirmed the RAPD-PCR analysis and microbiological tests. Sequencing of eight samples after amplification with specific forward 16S rDNA primers showed that five samples were Enterococus fecalis, one sample was Ruminococcus and two samples were uncultured bacteria, when compared with gene bank. Comparison of nucleotide sequence of 16S rDNA with similar sequence of bacterial isolates in gene bank, showed that there is similarity between them. Introduction The 16S rRNA gene is widely used as target as it is a highly conserved gene, ubiquitous in all organisms and contains variable and hypervariable regions of sequence. Molecular methods based on this gene are well established as a standard method for characterisation and identification of bacteria