Copyright © 2022 Mehmet Bahadır Çetinkaya and Hakan Duran.Biomedical image analysis based on metaheuristic algorithms is one of the most important research areas encountered in recent years. Due to the low contrast differences between the diseased areas and the image background in high-contrast biomedical images, effective methods are required to diagnose diseases with high accuracy. To overcome the difficulties encountered in this field, metaheuristic approaches may offer effective solutions due to their advantages such as the ability of converging to the global optimum, higher convergence rate, and having few control parameters. In this work, Jellyfish Search (JS), Marine Predators (MPA), Tunicate Swarm (TSA), Mayfly Optimization (MA), Chimp Optimization (ChOA), Slime Mould Optimization (SMA), Archimedes Optimization (AOA), and Equilibrium Optimizer (EO) algorithms, which are the most recently proposed metaheuristic algorithms in the literature, have been improved as clustering based in order to achieve vessel segmentation with high precision. Also, a detailed performance comparison of these algorithms has been realized for the rate of convergences, error values reached, CPU time, standard deviation, sensitivity, specificity, accuracy, F-score, and Wilcoxon rank sum-test. In order to present the compatibility of the results obtained with the literature, the performances of these novel algorithms have also been compared to that of Particle Swarm Optimization (PSO), Grey Wolf Optimization (GWO), and Differential Evolution (DE) algorithms. The simulation results represent that each algorithm produces similar convergence and error performance. Also, it can be emphasized from the statistical analyses that the stability and robustness of each metaheuristic approach are quite adequate in separating the vessel pixels and the background pixels of a retinal image. In general, this paper proves that although having fewer number of control parameters, the JS, MPA, TSA, MA, ChOA, SMA, AOA, and EO algorithms produce similar but a bit better results in terms of image segmentation when compared to PSO, GWO, and DE algorithms.