There are numerous factors that can have an impact on the microbial ecology and quality of frozen Pangasius hypophthalmus fillets during processing in Vietnam. The presence of spoilage bacteria along the processing line can shorten the shelf-life of thawed frozen fish products. Therefore, the spoilage microbiota throughout the processing chain of two companies (BC: large scale factory, chlorine-based process, BW: large scale factory, water-based process and SC: small scale factory, chlorine-based process) was identified by culture-dependent techniques and 16S rRNA gene sequencing. The microbiological counts were observed to be insignificantly different (p > 0.05) between BC and BW. Surprisingly, chlorine water treated fillets from the SC line were revealed to have significantly higher microbial counts than potable water treated fillets at BW line. This was determined to be a result of temperature abuse during processing at SC, with temperatures even greater than 10°C being recorded from skinning onwards. On the contrary, the microbiota related to spoilage for BC and BW lines was determined by 16S rRNA gene sequencing to be more diverse than that on the SC line. A total of 174 isolates, 20 genera and 38 species were identified along the processing chains. The genera Aeromonas, Acinetobacter, Lactococcus and Enterococcus were prevalent at various processing steps on all the processing lines evaluated. A diverse range of isolates belonging to the Enterobacteriaceae such as Providencia, Shigella, Klebsiella, Enterobacter and Wautersiella were isolated from fillets sampled on the SC line whereas Serratia was only observed on fillets sampled on the BC and BW lines. The results can be used to improve Good Manufacturing Practices for processed Pangasius fillets and to select effective measures to prolong the shelf-life of thawed Vietnamese Pangasius fillets products.