Two bacterial strains, Am4 and G10 were isolated from rumen fluid of different ruminants: cow (Holstein-Friesian) and sheep (Slovenskè merino), respectively. They were isolated from different hosts and regions, but showed 99.2% similarity of the 16S rRNA genes. Both strains are versatile and ferment various sugars to mainly succinate and lactate and small amounts of acetate and formate. The 16S rRNA sequences of Am4 and G10 revealed that they belonged to the genus Actinomyces, and are related to Actinomyces ruminicola JCM 13352 with 97.0% and 97.4% similarity, respectively. DDH showed strain Am4 and G10 had only 55.8 and 43.3% similarity with the Actinomyces ruminicola JCM 13352, and had 69.9% similarity among each other. Comparing strain Am4 and G10, gANI value and dDDH were 92.9% and 68.6%, respectively. Additionally, AAI between the strains was 95.8%. MLSA of housekeeping genes showed difference of metG and pheS. The G+C% contents of strain Am4 and G10 were 69.8% and 68.5%, respectively. MK-10(H) was the principal quinone for strain Am4 (82%) and G10 (91%) with small amounts of MK-10(H) and MK-10(H) for both strains. Only MK-9(H) was detected in strain Am4. MALDI-TOF analysis of protein profiles also revealed that Am4 and G10 are different from each other and from Actinomyces ruminicola JCM 13352. Based on phylogenetic and physiological characteristics, together with genome comparison and MLSA we propose two novel species in the genus Actinomyces: Actinomyces succiniciruminis sp. nov. (type strain Am4=TISTR 2317=DSM 10376) and Actinomyces glycerinitolerans sp. nov. (type strain G10=TISTR 2318=DSM 10377).