Background: Cardiovascular diseases (CVDs) are a leading cause of global morbidity and mortality, with ischemic heart disease (IHD) being the most common contributor. Acute myocardial infarction (AMI), a severe manifestation of IHD, requires timely treatment to restore blood flow to the myocardium. Streptokinase, a thrombolytic agent, is commonly used to dissolve clots and improve myocardial perfusion. However, thrombolytic failure, defined as insufficient ST-segment resolution on electrocardiogram (ECG) post-therapy, remains a major concern and is associated with adverse outcomes. To determine the frequency of thrombolytic failure with streptokinase in patients with AMI using ECG criteria and to identify factors associated with such failure. Methodology: This cross-sectional study was conducted at the Department of General Medicine, Lady Reading Hospital (LRH), Peshawar, over six months. A total of 149 patients aged 30–60 years diagnosed with AMI were enrolled using non-probability consecutive sampling. Patients with non-ST elevation myocardial infarction (NSTEMI), contraindications to streptokinase, prior streptokinase use, or symptom-to-needle time exceeding 24 hours were excluded. Two ECGs were performed—before streptokinase administration and 90 minutes after therapy. Thrombolytic failure was defined as less than 50% resolution in ST-segment elevation. Results: The mean age of participants was 45 ± 8.2 years, with 60% males and 40% females. The frequency of thrombolytic failure was 55%. Patients experiencing failure had a significantly longer mean symptom-to-needle time (7.1 ± 1.6 hours) compared to those with successful thrombolysis (5.4 ± 1.4 hours, p = 0.02). No significant associations were observed with age or gender. Secondary outcomes included an in-hospital mortality rate of 5%, recurrent myocardial infarction in 10%, and bleeding complications in 2.7%. Conclusion: Thrombolytic failure with streptokinase remains a significant challenge in AMI management, particularly with delays in therapy initiation. Strategies to reduce symptom-to-needle time and explore adjunctive therapies are crucial to improving outcomes.