Potato (Solanum tuberosum L.) is one of the emerging staple crops in Pakistan, with Punjab producing over 95% of the country’s potatoes. Wilt is an emerging threat to the potato crop worldwide, including in Pakistan. We identified and characterized Fusarium species associated with potato wilt in Pakistan through morphological and molecular analyses. Samples were collected during the 2020–2022 potato seasons from five major potato-growing regions: Sahiwal, Chichawatni, Pakpattan, Kamalia, and Faisalabad. Morphological characterization, internal transcribed spacer (ITS) sequencing, specific translation elongation factor 1-alpha (TEF) sequencing, and phylogenetic analysis were used to identify six different Fusarium species: F. oxysporum, F. equiseti, F. incarnatum, F. fujikuroi, F. annulatum and F. thapsinum. Pathogenicity tests in a greenhouse revealed that F. oxysporum and F. equiseti were responsible for Fusarium wilt in all sampled regions, with F. oxysporum being more prevalent in wilted samples. This is the first report of F. equiseti on wilted potatoes in Pakistan. In vitro biocontrol tests using Trichoderma harzianum showed 89% inhibition against F. equiseti and 65% inhibition against F. oxysporum. These findings on F. equiseti will aid in developing future control strategies, including biocontrol measures for Fusarium wilt in potatoes.