The study of Pestivirus bovis, formerly known as Bovine Viral Diarrhea Virus (BVDV), is of significant importance due to the economic challenges it poses in cattle herds in several countries. This virus leads to decreased productivity, reproductive failures, and increased susceptibility to secondary infections. The current study aimed to investigate the role of BVDV in abortion and infertility among cattle in Egypt using advanced diagnostic techniques. We employed a one-step multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) to detect BVDV, and the results showed an overall prevalence of 11.2% among 178 tested samples. Notably, 11.2% tested positive for BVDV-1, with higher detection rates in adult cows (12.1%) compared to calves (9.85%). Importantly, 13.8% of samples from dams with subfertility and repeated abortions were positive for BVDV-1. The results of rRT-PCR guided virus isolation using Madin–Darby bovine kidney (MDBK) cells. Identifying cytopathic effects (CPEs) in 15% of the samples, consistent with cytopathic-BVDV (CP-BVDV). An indirect fluorescent antibody assay (IFA) confirmed the presence of CP-BVDV in these samples. For non-cytopathic (NCP)- strains, the immunoperoxidase (IP) test using bovine turbinate (BT) cells was more effective, detecting NCP-BVDV in samples without CPE in MDBK cells. The presence of mixed infection was indicated by an isolate from a diarrheic calf, showing positive results for both CP-BVDV and NCP-BVDV. Plaque assays further confirmed CP-BVDV and NCP-BVDV isolation from mixed infections, highlighting the selective amplification of both biotypes. These findings underscore the significant role of BVDV-1 in reproductive disorders in cattle and the importance of employing comprehensive diagnostic methods for effective control and management strategies. Our study provides valuable insights that can guide future studies and improve herd health and productivity.