When I first started playing RE4, I knew nothing about the game, nothing at all. So when I killed Mendez and got out of the village gates I actually thought we were about to be rescued and that Leon would be sent off on another mission somewhere else. Yes I saw the castle up ahead but still it didn't dawn on me. Even the three ganados and the crazy truck didn't sway me. It wasn't until we passed the tipped over truck and then about twelve of those ganados came bursting through the gate, accompanied by the "Hard Road to Castle" music (one of the creepist in the game if you ask me) that I began to sense that the nightmare was just beginning, that far from being about to be rescued, it was about to get a whole lot worse...honestly, something about that particular "Hard Road to Castle" music screams "you're in for a hell of a ride yet, dude".
So yeah, for me, that first time on the road from the village to the castle, when the ganados burst out of nowhere, was one of the scarier moments in the game for me.
Apart from that, scariest moments were probably:
-When I was first holed up inside the double story house in Ch 1-1 in the village with Dr Salvador revving his chainsaw outside. Needless to say Leon lost his head that time...
-When I first ran into Colmillos near the lake at night in Ch 2-1...but I entered that area from the swamp area rather than from the dock area...at least when you come from the dock you get a cutscene that warns you of their presence...when you come from the swamp they jump on you the second you come through the door...that scared the crap out of me.
-When I first entered the Novistador sewer in Ch 3-2 and heard the thud and running footsteps of the Novistador but couldn't see a thing...
-When I entered the room after the clocktower fight in Ch 4-1 and saw TWO Garradors for the first time. Though I had fought two Garradors by that stage and so had some idea of how to fight them, seeing two in the same room was enough to make my eyes go wide. Very wide.
-When Oven Man burst out and decided to hug me...always a sure way to make a first timer jump in shock.
-After I had killed the first Regenerator, going back out into the corridor and hearing that tell tale breathing of more Regenerators....
-And finally, when I entered that freezer room with six frozen Regenerators...I remember I agonised over whether or not to turn off the cryogenic device...I had this vision that doing so might awaken six of the beasts at once! Little did I know then that only one awakened and only after taking the infrared scope...