Abstract Background Several researches have inspected the impact of inhalation anesthetics, intravenous anesthetics, and muscle relaxants on spectral entropy, but many didn’t evaluate the extent of neuromuscular block. Besides, they didn’t inspect the impact of distinct degrees of neuromuscular blockade on spectral entropy under dissimilar saturations of isoflurane inhalation. Hence, this study evaluated variant degrees of minimum alveolar concentration (MAC) to estimate the isoflurane concentration, along with various level of neuromuscular blockade. Aim of this study is to evaluate the effect of muscle relaxants (atracurium and rocuronium) on entropy readings (state, response entropy and response- state difference) during isoflurane anesthesia. This is a prospective randomized study where forty patients were included and divided into two study groups; patients in Group A received atracurium, while patients in Group R received rocuronium. State and response entropy were observed under MAC 0.8% and 1% under 50% and 100% neuromuscular blockage. Results There was a positive correlation between state (SE) and response entropy (RE) at baseline, different MAC, and different train of four (TOF) for both atracurium and rocuronium. State and response entropy decreased with increasing MAC of isoflurane (p < 0.001), while atracurium and rocuronium at TOF 50% and 100% showed no effect on SE or RE or RE-SE (P > 0.05). Conclusions State and response entropy can be used effectively to evaluate depth of anesthesia at different isoflurane MAC and atracurium or rocuronium doses. Registration number: ClinicalTrials.gov Identifier: NCT 05097508, Registered 5 October 2021 - prospectively registered, http://www.ClinicalTrial.gov